Polypeptides having interferon activity

ABSTRACT

A polypeptide having interferon activity which has an amino acid sequence corresponding to an amino acid sequence of an interferon selected from rIFN-α, hybrid rIFN-α, rIFN-β and rIFN-γ interferons in which at least one cysteine residue in the amino acid sequence has been replaced by an amino acid residue which is incapable of forming intermolecular disulfide bonds is provided. There are also provided a dsDNA sequence encoding the novel polypeptide; a replicable plasmidic expression vehicle containing the dsDNA encoding the novel polypeptides of the invention; a microorganism which has been transformed with the replicable plasmidic expression vehicle; and a method of preparing the dsDNA which encodes the novel polypeptides of the invention.

This is a division of application Ser. No. 06/827,674, filed Feb. 10,1986 now U.S. Pat. No. 4,816,566, which is a continuation of Ser. No.499,964, filed June 1, 1983, now abandoned.

BACKGROUND OF THE INVENTION

Interferons are proteins which are produced by a number of differentkinds of organisms and which are presently grouped into three majorclasses, designated leukocyte interferon (IFN-α), fibroblast interferon(IFN-β) and immune interferon (IFN-γ). The interferons have antiviraland antiproliferative activities, a potent ability to confer avirus-resistant state in targeted cells and immunomodulatory activities.Their biological properties have led to the clinical use of interferonsas therapeutic agents for the treatment of viral infections andmalignancies.

Interferons have been produced from natural sources such as buffy coatleukocytes and fibroblast cells, optionally using known inducing agentsto increase the production of interferon. Interferons have also beenproduced by recombinant DNA techniques, i.e. by expression from amicroorganism which has been transformed with an expression vectorcontaining an interferon gene under the control of a promoter-operatorsequence. (Leukocyte, fibroblast and immune interferons produced byrecombinant techniques are designated rIFN-α, rIFN-β and rIFN-γ,respectively). As many as 12 distinct genes encoding for differentspecies of rIFN-α have been cloned. These various species are designatedrIRN-αA, rIFN-αB, rIFN-αC and so forth.

Goeddel and coworkers achieved the initial expression of rIFN-αA in E.coli cells containing the recombinant plasmid pL 31. (Nature, 287, 411(1980)). This plasmid contains the structural gene for mature rIFN-αA(i.e., a gene in which the nucleotide sequence encoding a 23-amino acidsignal peptide normally translated in the human cell has been removedand an ATG "start" signal has been inserted immediately before the codonfor the first amino acid following the signal peptide) under the controlof an appropriately positioned promoter-operator sequence. The rIFN-αAproduced in this manner has been employed in the clinical treatment ofpatients suffering from a variety of viral and neoplastic diseases.

The rIFN-α interferons are 165 amino acids (in the case of rIFN-αA) or166 amino acids in length, except that they may, in some instancescontain a methionine attached to the N-terminus of the oridinarily firstamino acid of the protein as the result of translation of the ATG startsignal which encodes methionine.

Hybrid leukocyte interferons have been produced by expression of geneswhich are produced by cleaving two or more genes encoding differentleukocyte interferons at internal endonuclease cleavage sites and thenligating one or more cleavage fragments of one gene with one or morecleavage fragments of a different gene (or genes) to produce a geneencoding a complete 165- or 166-amino acid leukocyte interferon havingone or more segments corresponding to a portion of a first leukocyteinterferon species and one or more segments corresponding to portions ofdifferent leukocyte interferon species. In this manner, for example, ithas been possible to produce a leukocyte interferon in which the aminoacid sequence corresponds to that of rIFN-αA at positions 1-92 and tothat of rIFN-αD at positions 92-166. Similarly, by ligating the genecleavage fragments in reverse order, it has been possible to produce aleukocyte interferon in which the amino acid sequence corresponds tothat of rIFN-αD at positions 1-92 and to that of rIFN-αA at positions93-165. (J. Biol. Chem., 257, pp. 11497-11502 (1982)).

A problem which has occurred in the manufacture and use of interferonsis that the individual interferon molecules tend to oligomerize. Theetiology of these oligomers has not been completely understood. It isbelieved, however, that the procedures used to purify interferons fortherapeutic use may contribute to the oligomerization problem. Presentlyavailable purification methods, such as high pressure liquidchromatography or monoclonal antibody affinity chromatography arecarried out under conditions which can favor the formation of dimers,trimers and higher oligomers of interferon. These oligomeric forms ofinterferon result from two or more interferon molecules becomingirreversibly associated with one another through intermolecular covalentbonding, such as by disulfide linkages. This problem has been observedparticularly with respect to leukocyte and fibroblast interferons.

While the dimeric forms of interferons are believed to retain biologicalactivity, the higher oligomeric forms in many cases have either nobiological activity or significantly reduced activity by comparison tothe monomeric forms. Moreover, the oligomeric forms have the potentialfor causing deleterious side effects if used therapeutically.

All of the known rIFN-α, rIFN-β and rIFN-γ interferons contain multiplecysteine residues. These residues contain sulfhydryl side-chains whichare capable of forming intermolecular disulfide bonds, which result inoligomerization, as well as intramolecular disulfide bonds. The aminoacid sequence of rIFN-αA contains cysteine residues at positions 1, 29,98 and 138. Wetzel and coworkers assigned intramolecular disulfide bondsbetween the cysteine residues and positions 1 and 98 and between thecysteine residues and positions 29 and 138. Nature, 289, 606 (1981).

Because of the importance of eliminating or preventing the occurrence ofoligomers in interferon preparations, considerable efforts have beenexpended to overcome the oligomerization problem. Heretofore, effortshave been concentrated on adjusting the purification conditions toprevent the formation of oligomers or post-processing with reagents andreaction conditions which reduce intermolecular disulfide bonds.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to novel proteins which display the biologicalactivities of the known interferons without the concomitant problem ofoligomerization that is associated with the known interferons. Inparticular, we have prepared novel polypeptides which correspond inamino acid sequence to known interferons, except that certain cysteineresidues have been replaced by residues of other amino acids which areincapable of forming disulfide bonds. Quite surprisingly, we have beenable to make these amino acid substitutions without any diminution inthe biological activity of the resultant protein as compared to that ofthe known interferon from which it was derived.

In one broad aspect, this invention relates to a novel polypeptidehaving interferon activity comprising an amino acid sequencecorresponding to an amino acid sequence of an interferon selected fromrIFN-α, hybrid rIFN-α, rIFN-β and rIFN-γ interferons in which at leastone cysteine residue has been replaced by an aminoacid residue which isincapable of forming intermolecular disulfide bonds, i.e. an amino acidresidue other than cysteine.

In a preferred embodiment of this broad aspect, the invention relates toa polypeptide comprising an amino acid sequence corresponding to anamino acid sequence of rIFN-αA in which at least one of the cysteineresidues at positions 1 and 98 has been replaced by an amino acidresidue which is incapable of forming intermolecular disulfide bonds.

This broad aspect of the invention also relates to antiviralcompositions comprising the novel polypeptides, wherein the compositionis essentially free of oligomers of higher aggregation than the dimericform and in one preferred embodiment, contains only stable monomericinterferon.

In a second broad aspect, this invention relates to methods andintermediates for producing the novel polypeptides described above bythe techniques of DNA recombination. In particular, this aspect relatesto a method of producing a double stranded DNA (dsDNA) encoding thenovel polypeptide by using restriction enzymes to excise a portion ofthe dsDNA encoding for the undesired cysteine residue in the parentalinterferon gene and replacing it with a synthetic oligodeoxynucleotidesegment in which the nucleotide triplet encoding the cysteine residuehas been replaced by a nucleotide triplet encoding an amino acid residuewhich is incapable of forming intermolecular disulfide bonds.

Accordingly, this second broad aspect of the invention encompasses dsDNAwhich encodes the novel polypeptides of the invention; a replicableplasmidic expression vehicle containing the dsDNA encoding the novelpolypeptides of the invention; a mircoorganism which has beentransformed with the replicable plasmidic expression vehicle; and amethod of preparing the dsDNA which encodes the novel polypeptides ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the amino acid sequence of rIFN-αA and anucleotide sequence which encodes rIFN-αA.

FIG. 2 is a schematic representation of the process used to produce thedsDNA encoding rIFN-αA (Gly 1).

FIG. 3 is a schematic representation of the process used to produce thedsDNA encoding rIFN-αA (Gly 1/Ser 98).

FIG. 4 is a schematic representation of the process used to produce thereplicable plasmidic expression vehicle for expression of rIFN-αA (Gly1).

FIG. 5 is a photographic representation of a sodium dodecylsulfatepolyacrylamide gel on which rIFN-αA and rIFN-αA (Gly 1) wereelectrophoresced for detection of oligomers.

FIG. 6 is a schematic representation of the process used to prepare thereplicable plasmidic expression vehicle for expression of rIFN-αA (Gly1/Ser 98).

FIG. 7 is a photographic representation of a sodium dodecylsulfatepolyacrylamide gel on which rIFN-αA (Gly 1) and rIFN-αA (Gly 1/Ser 98)were electrophoresced for detection of oligomers.

Asterisks in the figures indicate nucleotide substitutions replacing thenucleotide triplets encoding cysteines.

DETAILED DESCRIPTION OF THE INVENTION I. The Novel Polypeptides andCompositions

As previously indicated, the invention relates to a novel polypeptidehaving interferon activity comprising an amino acid sequencecorresponding to an amino acid sequence of an interferon selected fromrIFN-α, hybrid rIFN-α, rIFN-β and rIFN-γ interferons in which at leastone cysteine residue in the amino acid sequence has been replaced by anamino acid residue which is incapable of forming intermoleculardisulfide bonds, i.e. a residue other than cysteine.

The amino acid structures of the parental interferons which can bemodified by replacement of cysteine residues to produce the novelpolypeptides are known in the art. The cloning and expression of rIFN-αAwas described by Goeddel and coworkers in Nature, 287, 411 (1980). FIG.1 gives the amino acid sequence of mature rIFN-αA. The line above theamino acid sequence gives the associated nucleotide sequence of a codingstrand of dsDNA which encodes the mature rIFN-αA. Other species ofrIFN-α's can also be modified to produce the novel polypeptides of theinvention. The amino acid sequences of rIFN-αA, B, C, D, F, G, H, K andL, as well as the nucleotide sequences which encode them are describedby Pestka in Archiv. Biochem. Biophys., 221, 1 (1983). The amino acidsequences and associated nucleotide sequences of rIFN-αE, I and J aredescribed in British Patent Specification No. 2,079,291, published Jan.20, 1982.

The novel polypeptides of the invention can also be produced bymodifying the amino acid structure of a hybrid rIFN-α. As used herein,the term "hybrid rIFN-α" refers to a leukocyte interferon derived from atransformant microorganism by the expression of a gene which encodes aleukocyte interferon in which one or more segments of the moleculecorrespond to a segment of a first leukocyte interferon species and oneor more other segments correspond to segments of different leukocyteinterferon species. Exemplary of such hybrids are hybrids of rIFN-αA andrIFN-αD which are disclosed by Pestka in Archiv. Biochem. Biophys., 221,1 (1983).

The cloning and expression of mature rIFN-β is described by Goeddel etal. in Nucleic Acids Research, 8, 4057 (1980). The amino acid structure,as well as the nucleotide sequence which encoded the amino acidstructure, are given in the publication.

The cloning and expression of mature rIFN-γ are described by Gray, P. W.et al., in Nature, 295, 503 (1982). The amino acid structure and thenucleotide sequence which encoded the amino acid sequence are given.

The novel polypeptides of this invention can also be prepared by themodification of parental interferons which represent allelic variationsof the specific rIFN-α, hybrid rIFN-α, rIFN-β and rIFN-γ interferons ofthe publications cited in the preceding paragraphs. For example, Nagataand coworkers described the expression of an rIFN-α gene (denoted asrIFN-α₁) which expressed a polypeptide (in non-mature form) whichdiffers from rIFN-αD by a single amino acid at position 114. (Nature,284, 316 (1980)). Similarly the cloning and expression of an rIFN-α gene(identified as rIFN-α₂) which expressed a polypeptide differing fromrIFN-αA by a single amino acid at position 23 was described in EuropeanPatent Application No. 32 134, published July 15, 1981. DNA sequencesencoding these and other allelic variations of the parental rIFN's canbe modified to encode the novel polypeptides of this invention, providedthat the parental rIFN contains a cysteine residue which is not requiredfor biological activity and which is capable of participating in theformation of intermolecular disulfide bonds.

Preferred polypeptides of the invention are those obtained bysubstitution of cysteine residues in rIFN-α's or hybrid-α's. All therIFN-α's, and consequently all hybrid rIFN-α's, contain cysteineresidues at the N-terminus, i.e. at position 1, and at position 29,except for rIFN-αE, which does not have cysteine at position 29.Additionally, rIFN-αA contains cysteine residues at positions 98 and138; rIFN-αB contains cysteine residues at positions 100 and 139;rIFN-αC, F, G, H, I, J, K and L contain cysteine residues at positions99 and 139; and rIFN-αD contains cysteine residues at positions 86, 99and 139. We note that Pestka, in Archiv. Biochem. Biophys., 221, 1(1983), shows cysteine residues at positions 99 and 139 of rIFN-αA inFIG. 17 of the article, rather than at positions 98 and 138. This isbecause the sequence representation has been shifted one positionfollowing position 43 to align it with the other rIFN-α species whichcontain one more amino acid residue than rIFN-αA. All amino acidpositions recited herein refer to sequentially numbered amino acids anddo not account for such shifts.

The polypeptides of the invention have amino acid sequencescorresponding to amino acid sequences of known rIFN's in which cysteineresidues have been replaced by amino acid residues which are incapableof forming intermolecular disulfide bonds. The cysteine residues whichare replaced are those which are not required for the rIFN to maintaininterferon activity. As used herein the term "interferon activity"refers to the characteristic antiviral and antigrowth activitycharacteristic of the interferons. The characteristic antiviral activityof rIFN can be determined using the cytophatic effect inhibition testdescribed in Familleti, P. C. et al., Methods in Enzymology 78, 387(1981). The characteristic antigrowth activity of rIFN can be determinedusing the procedure described in Evinger, M. & Pestka, S., Methods inEnzymology, 79, 45 (1981).

Since the intramolecular disulfide bridge between the cysteine residuesat positions 29 and 138 of rIFN-αA is believed to be required forinterferon activity, we have left these residues intact. Similarly, thecysteine residues at positions 29 and 139 of rIFN-αB through L (orallelic variations thereof) are not replaced.

It has also been shown by Shepard et al. (Nature, 294, 563 565 (1981))that substitution of the cysteine at position 141 of rIFN-β resulted ina loss of biological activity. Accordingly, this cysteine resiude is notto be replaced.

We have replaced the cysteine residue at position 1 of rIFN-αA, and wehave replaced both cysteine residues at positions 1 and 98 of rIFN-αA,without any concomitant loss of interferon activity. Analogously, thecysteine residues at position 1 and/or position 99 (in the case ofrIFN-αC through L) and 100 (in the case of rIFN-αB) can be replaced. Inthe case of a hybrid rIFN-α, cysteine residues may be replaced atposition 1 and/or position 98, 99 or 100, depending upon which parentalrIFN corresponds to that segment of the hybrid rIFN-α.

In the case of rIFN-αD or a hybrid rIFN-α in which a portion of theamino acid sequence corresponds to a portion of the amino acid sequenceof rIFN-αD which contains position 87, the cysteine residue at position87 may also be replaced.

The amino acid residues which replace the cysteine residues of theparental interferons in the polypeptides of the invention can be anyamino acid residues which are incapable of participating in theformation of intermolecular disulfide bonds; that is, any amino acidresidues other than cysteine. In the case of the N-terminal cysteineresidue, i.e. the residue at position 1, we prefer to replace this withglycine, since glycine does not have a reactive side chain. In the caseof the internal cysteine residues, eg. the residue at position 98 ofrIFN-αA, we prefer to replace these with serine, since the serine sidechain most closely resembles the spatial arrangement of the cysteineside chain. Thus, substitution with serine presents the leastpossibility for disrupting the conformation of the molecule.

We note that the rIFN-α, hybrid rIFN-α, and rIFN-γ interferons possess acysteine residue at position 1. If desired, one can produce a novelpolypeptide having interferon activity by deleting this cysteineentirely. This can be done by excising a portion of the gene for theparental interferon which encodes the N-terminal cysteine, in a mannersuch as that described in Example 1 herein and replacing it with asynthetic oligodeoxynucleotide corresponding to the excised portion ofthe gene except that the nucleotide triplet encoding the cysteine hasbeen deleted; that is, the ATG translation initiation signal isimmediately followed by the nucleotide triplet encoding the amino acidresidue at position 2 of the parental interferon. The resultant dsDNAcan be incorporated into an expression vehicle which can be used totransform a microorganism. The transformant microorganism can then begrown up and used to express a novel polypeptide having interferonactivity which comprises an amino acid sequence corresponding to theamino acid sequence of an rIFN-α, hybrid rIFN-α, or rIFN-γ interferon inwhich the cysteine residue at position 1 has been deleted.

In a preferred embodiment of the invention, we have modified a geneencoding rIFN-αA (the amino acid sequence of which is given in FIG. 1),in a manner described in detail hereinafter, and expressed the gene toproduce a polypeptide in which one or both cysteines at positons 1 and98 have been replaced by amino acid residues which are incapable offorming intermolecular disulfide bonds. Accordingly, a preferredembodiment of our invention is a polypeptide of the formula ##STR1##wherein at least one of R¹ and R² is an amino acid residue having a sidechain which is incapable of participating in the formation of anintermolecular disulfide bond and the other is cysteine or an amino acidhaving a side chain which is incapable of participating in the formationof an intermolecular disulfide bond.

Initially, we modified the gene for rIFN-αA by excising a portion of thegene which included the nucleotide triplet encoding cysteine at position1 and replacing it with a synthetic oligodeoxynucleotide that encodedglycine at position 1. The section of the gene containing the nucleotidetriplet for the cysteine at position 98 was unchanged. The modified genein a plasmidic expression vehicle, was expressed in transformed E. colito produce a polypeptide whose amino acid sequence corresponded to theamino acid sequence of rIFN-αA except that it contained a glycine atposition 1 (the modified polypeptide is referred to herein as rIFN-αA(Gly 1)). After extraction from the cells and purification on amonoclonal antibody immunoaffinity chromatography column, the interferonthus obtained was subjected to electrophoresis on sodium dodecylsulfatepolyacrylamide gel. Upon electrophoresis the interferon separated into amajor band, corresponding to nomomeric material, and a minor band,corresponding to dimeric material. An rIFN-αA expressed from theunmodified gene for rIFN-αA was electrophoresced on the same gel andseparated into bands corresponding to monomer, dimer and oligomers ofhigher aggregation than dimer, e.g trimer, tetramer, etc

The composition containing a major proportion (more than half) ofmonomeric rIFN-αA (Gly 1) and a minor proportion of dimeric rIFN-αA(Gly 1) displayed the full antiviral activity characteristic of rIFN-αAon MDBK cells.

The gene containing the synthetic oligodeoxynucleotide segment encodingglycine at position 1 was further modified by excising a segment of thegene encoding cysteine at position 98 and replacing it with a syntheticoligodeoxynucleotide that encoded serine at position 98. This gene, in aplasmidic expression vehicle, was expressed in transformed E. coli toproduce a polypeptide whose amino acid sequence corresponded to theamino acid sequence of rIFN-αA except that it contained glycine atposition 1 and serine at position 98. (The modified polypeptide isreferred to herein as rIFN-αA (Gly 1/Ser 98)). After extraction fromcells and purification by monoclonal antibody immunoaffinitychromatography column, the interferon thus obtained was subjected toelectrophoresis on sodium dodecylsulfate polyacrylamide gel. Theinterferon migrated in a single band, indicating essentially onlymonomeric material.

The composition comprising essentially all monomeric rIFN-αA (Gly 1/Ser98) displayed antiviral activity which was about the same as that ofrIFN-αA on MDBK cells.

The novel polypeptides of the invention can be used for the samepurposes as the known interferons, eg. as antiviral and antitumor agentsand as a treatment for immunosupressive conditions. Dosage and dose ratemay parallel that currently being used in clinical applications of theknown interferons, typically about 1-200×10⁶ units daily. Thepolypeptides can be conveniently administered in parenteral dosage form.Suitable dosage forms can be prepared using known formulation methods toprepare pharmaceutically useful compositions wherein the polypeptide isadmixed with a pharmaceutically acceptable carrier vehicle. A suitabledosage form will comprise an effective amount of the polypeptidetogether with a pharmaceutically acceptable carrier vehicle suitable forthe particular host and mode of administration

II. Recombinant DNA Methods and Intermediates

As previously indicated, the novel polypeptides of this invention can beprepared, using the techniques of DNA recombination, by modifying a genewhich encodes an rIFN-α, hybrid rIFN-α, rIFN-β or rIFN-γ to produce agene which encodes the novel polypeptide; incorporating the modifiedgene into a replicable plasmidic expression vehicle; transforming a hostmicroorganism with the expression vehicle containing the modified gene;growing up the transformed microorganism; expressing the polypeptide inthe microorganism; and recovering the expressed polypeptides. One ormore portions of the gene encoding the parental interferon, e.g.rIFN-αA, which contain the nucleotide triplets encoding the cysteineresidues which are to be replaced, are excised and replaced withsynthetic oligodeoxynucleotides encoding the desired sequence(s) havingamino acid residues which are incapable of forming intermoleculardisulfide bonds.

In a preferred embodiment of the invention, a gene encoding rIFN-αA(which is illustrated in FIG. 1) is modified to produce a doublestranded DNA comprising a coding strand and a complementary strand,wherein the coding strand, reading from the 5' end comprises thesequence ##STR2## wherein at least one of X and Y is a nucleotidetriplet encoding an amino acid residue which has a side chain that isincapable of forming an intermolecular disulfide bond and the other is anucleotide triplet encoding cysteine or an amino acid residue which isincapable of forming an intermolecular disulfide bond.

In particular, we have provided a method of producing a double strandedDNA encoding a polypeptide having interferon activity which comprises:

(a) cleaving a double stranded DNA which contains a sequence encoding aninterferon selected from rIFN-α, hybrid rIFN-α, rIFN-β and rIFN-γinterferons by endonuclease cleavage to prouce a first cleavage fragmentcontaining a nucleotide triplet encoding a cysteine residue and one ormore other cleavage fragments encoding the remainder of the interferon;

(b) separating the cleavage fragment containing the nucleotide tripletencoding the cysteine from the cleavage fragments encoding the remainderof the interferon;

(c) preparing a double stranded oligodeoxynucleotide sequencecorresponding to the separated cleavage fragment in which the nucleotidetriplet encoding said cysteine residue has been replaced by a nucleotidetriplet encoding an amino acid residue which is incapable of formingintermolecular disulfide bonds, said double strandedoligodeoxy-nucleotide having ends complementary to the ends of thedouble stranded DNA encoding the remainder of the interferon; and

(d) ligating said double stranded oligodeoxynucleotide to the doublestranded DNA encoding the remainder of the interferon in the properorientation for the expression of the polypeptide.

As used herein the term "upstream" refers to the direction moving towardthe 5' end of the coding strand and the term "downstream" refers to thedirection moving toward the 3' end of the coding strand.

The dsDNA encoding an rIFN which is cleaved in step (a) above can beconveniently obtained from an expression vector or cloning vectorcontaining the structural gene. As a source of dsDNA to serve as astarting material from which to prepare the dsDNA encoding rIFN-αA(Gly 1) and rIFN-αA (Gly 1/Ser 98), we employed plasmid pL 31 Thisplasmid, derived from pBR322, contains a 1160 base pair insert encodinga trp promoter-operator, ribosome binding site, ATG translationinitiation signal, a structural gene encoding the amino acid sequence ofrIFN-αA depicted in FIG. 1 and an untranslated 3' sequence following thestop codon (Goeddel et al., Nature, 287 411 (1980)).

The endonucleases which are used to cleave the dsDNA in step (a) arewell known to those familiar with the techniques of DNA recombination.They are restriction enzymes which selectively recognize and cleavecertain specific DNA sequences. The particular endonucleases used willdepend on the particular rIFN gene involved and the particular cysteineresidue(s) to be replaced. It is well within the skill level of theworker in the art to identify appropriate cleavage sites and to selectan appropriate cleavage endonuclease. The reaction conditions underwhich the cleavage reactions take place, as well as the methods forseparating and purifying the fragments, are well known in the art.

In preparing a dsDNA sequence encoding rIFN-αA (Gly 1), we tookadvantage of the existence of a Sau 3AI recognition site which cleavesthe structural gene for rIFN-αA immediately following the nucleotidetriplet of the coding strand which codes for cysteine at position 1 toseparate the nucleotide triplet encoding the undesired cysteine from theremainder of the structural gene.

The procedure which we employed to produce a dsDNA encoding rIFN-αA(Gly 1) can be described as follows, with specific reference to FIG. 2.The plasmid pL 31, which contained the structural gene for rIFN-αA (thecoding strand of which is depicted in FIG. 1), was digested completelywith Pst I and partially with Sau 3AI. An 854 base pair cleavagefragment was isolated. This fragment, beginning at the Sau 3AI sitecontained the full sequence of bases encoding rIFN-αA except for the TGTencoding cysteine at position 1 and terminated at the Pst I site 360bases beyond the stop codon on the coding strand.

The 5' end of the rIFN-αA gene thus removed was replaced by ligating the854 base pair segment with the synthetic oligodeoxynucleotide ##STR3##in which the --GGC-- encoding a glycine residue replaces the --TGT--encoding cysteine in the unmodified rIFN-αA gene. The 865 base pairdsDNA thus produced contained the sequence encoding rIFN-αA (Gly 1) andterminated at an Eco RI site at the 5' end of the coding strand and aPst I site at the other end.

In order to modify the rIFN gene to replace an internal cysteineresidue, e.g. the cysteine residue at position 98 of rIFN-αA or rIFN-αA(Gly 1), the dsDNA encoding the rIFN can be cleaved at a firstendonuclease cleavage site upstream of the nucleotide triplet encodingthe undesired cysteine and at a second endonuclease cleavage sitedownstream of the nucleotide triplet encoding the undesired cysteine toisolate a fragment containing the undesired nucleotide triplet. Thefragment containing the undesired nucleotide triplet is separated fromthe dsDNA encoding the remainder of the rIFN. A syntheticoligodeoxynucleotide is prepared which corresponds to the removedsegment except that the nucleotide triplet encoding cysteine is replacedby a nucleotide triplet encoding a different amino acid residue,preferably serine. This synthetic oligodeoxynucleotide is then ligatedin the proper orientation to the dsDNA encoding the remainder of therIFN.

If it is desired to delete two internal cysteine residues which aresufficiently close together, this may be done by cleaving the gene at afirst endonuclease cleavage site upstream of the nucleotide tripletencoding the first undesired cysteine residue and at a secondendonuclease cleavage site downstream of the nucleotide triplet encodingthe second undesired cysteine; and replacing the segment thus removedwith a synthetic oligodeoxynucleotide encoding a segment in which bothcysteines are replaced by different amino acids.

With reference to FIG. 3, we used the following procedure to produce adsDNA encoding rIFN-αA (Gly 1/Ser 98). The starting material employedwas an 869 base pair dsDNA encoding rIFN-αA (Gly 1). This was the 865base pair of dsDNA produced by the previously described procedure towhich an additional 4 base pairs had been ligated to the 3' end in orderto create a terminal Eco RI recognition site for insertion into anexpression vector. This dsDNA was cleaved with Pvu II, resulting in a273 base pair segment containing the 5' end of the coding strand forrIFN-αA (Gly 1) and a 596 base pair segment containing the 3' end of thecoding strand for rIFN-αA (Gly 1), which included the nucleotide tripletencoding cysteine at position 98. The 596 base pair segment waspartially digested with Hinf I to produce a 48 base pair fragmentcontaining the nucleotide triplet encoding the cysteine at position 98and a 548 base pair fragment encoding the remainder of the 3' end. Twosynthetic double stranded oligodeoxynucleotide were prepared to replacethe 48 base pair region. The synthetic double strandedoligodeoxynucleotides, which contained a 9-base overlapping region, hadthe following sequences: ##STR4##

The AGC nucleotide triplet encoding serine in Block II replaces the TGTencoding cysteine in the deleted 48 base pair fragment. Block I wasligated to the 273 base pair fragment and Block II was ligated to the548 base pair fragment of the gene. The resulting two fragments wereligated at the 9-base overlap to produce an 869 base pair dsDNAcontaining a coding strand which encoded rIFN-αA (Gly 1/Ser 98).

The dsDNA encoding the novel polypeptide of the invention can beincorporated into an expression vehicle which can be used to transform ahost microorganism for the purpose of expressing the novel polypeptide.Any of the known and commonly employed expression vectors may beemployed for this purpose, particularly plasmidic expression vectors. Aplasmid is a non-chromosomal loop of double stranded DNA found inbacteria and other microbes, often in multiple copies per cell. Includedin the information encoded in the plasmid DNA is that required toreproduce the plasmid in daughter cells (i.e. a "replicon") and,ordinarily, one or more selection characteristics such as, in the caseof bacteria, resistance to antibiotics which permit clones of the hostcell containing the plasmid of interest to be recognized andpreferentially grown in selective media.

A plasmidic expression vector must also incorporate the control elementsnecessary to regulate expression of the structural gene. Expression isinitiated in a region known as the promoter which is recognized by andbound by RNA polymerase. In some cases, as in the lambda phage or "P_(L)" promoter which we employed in the practice of the present invention,promoter regions are overlapped by "operator" regions to form a combinedpromoter-operator. Operators are DNA sequences which are recognized byso-called repressor proteins which serve to regulate the frequency oftranscription initiation at a particular promoter. The polymerasetravels along the DNA, transcribing the information contained in thecoding strand from its 5' to its 3' end into messenger RNA which is inturn translated into a polypeptide having the amino acid sequence forwhich the DNA encodes. Each amino acid is encoded by a nucleotidetriplet or "codon" within the structural gene, i.e. that part whichencodes the amino acid sequence of the expressed product. After bindingto the promoter, the RNA polymerase first transcribes nucleotidesencoding a ribosome binding site, then a translation initiation or"start" signal (ordinarily ATG, which in the resulting messenger RNAbecomes AUG), then the nucleotide codons within the structural geneitself. So-called stop codons are transcribed at the end of thestructural gene whereafter the polymerase may form an additionalsequence of messenger RNA which, because of the presence of the stopsignal, will remain untranslated by the ribosomes. Ribosomes bind to thebinding site provided on the messenger RNA, in bacteria ordinarily asthe mRNA is being formed, and themselves produce the encodedpolypeptide, beginning at the translation start signal and ending at thepreviously mentioned stop signal. The desired product is produced if thesequences encoding the ribosome bindng site are positioned properly withrespect to the AUG initiator codon and if all remaining codons followthe initiator codon in phase.

To incorporate the dsDNA encoding the novel polypeptide of the inventioninto the expression vector, the vector is first linearized by cutting itwith an endonuclease at a cleavage site which is appropriatelypositioned with respect to the promoter-operator sequence to allowexpression of the inserted structural gene. The dsDNA encoding the novelpolypeptide is then ligated at either end to the cleaved ends of thevector to recircularize the vector. Of course, the ends of the dsDNAinsert must be complementary to the cleaved ends of the vector to allowligation. If necessary, the dsDNA can be rendered complementary bybuilding up or cleaving back the ends, using known procedures, toprovide complementary ends, provided that the structural gene itself andthe associated initiation and termination codons are left intact andthat the gene remains in the proper position and reading frame withrespect to the promoter-operator sequence to allow expression.

Accordingly, there is provided, in accordance with the teachings of thisinvention, a replicable plasmidic expression vehicle comprising:

(a) a replicon;

(b) a promoter-operator sequence;

(c) a DNA sequence encoding a ribosome binding site; and

(d) a DNA sequence, in phase with said promoter-operator sequence,comprising a translation initiation signal, a sequence encoding apolypeptide having interferon activity, said polypeptide comprising anamino acid sequence of an interferon selected from rIFN-α, hybridrIFN-α, rIFN-β and rIFN-γ interferons in which at least one cysteineresidue in the amino acid sequence has been replaced by an amino acidresidue which is incapable of forming intermolecular disulfide bonds,and a translation termination signal.

Any of the known plasmidic expression vectors can be employed in thepreparation of the replicable plasmidic expression vehicle of thisinvention. We prefer to employ plasmid pRC 23, an expression vectorwhich is derived from pBR 322 and incorporates the P_(L)promoter-operator sequence (Bernard, H. U. and Helinski, D. R., Methodsin Enzymology, 68, 482). The vector pRC 23 is constructed by ligatingsynthetic oligodeoxynucleotides containing a "consensus" rebisomebinding site (Scherer et al., Nucleic Acids Research 8, 3895 (1980)) toa 250 bp Bgl II-Hae III fragment containing the P_(L) promoter andinserting the ligation product into the plasmid pRC 2. Further detailsconcerning the construction of the cloning and expression vector pRC 23can be obtained by reference to U.S. patent application Ser. No.397,388, filed July 12, 1982 entitled "Novel Vectors and Methods forControlling Interferon Expression", inventor Robert M. Crowl. In thisvector, the operator is recognized by a temperature-sensitive repressorprotein which is inactive at a temperature above about 36° C.; that is,it does not bind the operator at a temperature above 36° C. Therepressor protein is encoded by the cIts gene, which is carried on acompatible plasmid pRK 248. Thus transcription and expression of genecan be controlled by controlling the temperature at which therecombinant microorganism incorporating the expression vehicle ismaintained. Plasmic pRC 23 is designed to accept dsDNA sequences whichterminate in Eco RI recognition sites. Accordingly, the 869 base pairdsDNA sequences previously mentioned, which encode either rIFN-αA(Gly 1) or rIFN-αA (Gly 1/Ser 98) and terminate in Eco RI recognitionsites at either end, can be inserted directly into pRC 23 which has beenlinearized by cleavage with Eco RI. Ligation is carried out under knownconditions using a known ligase e.g. T4 DNA ligase.

The replicable plasmidic expresion vehicle is used to transform amicroorganism, preferably an E. coli, to produce a transformant which iscapable of expressing the polypeptide of the invention. Transformationcan be conveniently carried out using known procedures such as treatingthe host cells with CaCl₂ at about 4° C. to render the cell wallspermeable to the replicable plasmidic expression vehicle (see, e.g.Humphries et al., Transformation, eds. Glover & Butler, 287 312 (1979)).The transformed microorganism can be grown up under known fermentationconditions and the novel polypeptide expressed in the microorganism. Thepolypeptide is then recovered, for example, by lysing the cells torelease the polypeptide and purifying the polypeptide by knowntechniques. We prefer to purify the polypeptide by immunoaffinitychromatography on a column having monoclonal antibodies to thecorresponding parental rIFN bound to a solid support. A suitablemonoclonal antibody for use in purifying rIFN-αA (Gly 1) or rIFN-αA (Gly1/Ser 98) is described by Staehelin et al. in Journal of BiologicalChem., 256, 9750 (1981).

When the expression vector employed to produce the replicable plasmidicexpression vehicle is pRC 23, the resultant transformant microorganismcan be grown up to a desired density at a temperature of 30° C. and thetempperature can then be raised to about 42° C., to inactivate therepressor protein and initiate expression.

The following examples are presented in order to further illustrate thepractice of this invention and are not intended to limit the inventionin any way. Unless otherwise stated, all parts and percents are byweight and all temperatures are centigrade.

EXAMPLE 1 Preparation of rIFN-αA (Gly 1)

(a) Preparation of dsDNA Encoding rIFN-αA (Gly 1)

Plasmid pL 31 (6 μg) containing the gene encoding rIFN-αA, was digestedcompletely with Pst I and partially Sau 3AI. Both digests were performedat 37° C. using 20 units Pst I for 1 hr. followed by 5 units of Sau 3AIfor 5 min. in a 20 μl reaction. The cleavage fragments were separated in1.5% Agarose gel. An 854 base pair fragment was isolated which beganwith the nucleotide triplet encoding Asp at position 2 of rIFN-αA andterminated at the Pst I site 360 base pairs beyond the translationtermination codon.

Using the procedure of Miyoshi, et al., Nucleic Acids Res. 8, 5507-5517(1980), the synthetic oligodeoxynucleotides ##STR5## were prepared andthe 5' ends phosphorylated for ligation. The phosphorylatedoligodeoxynucleotides were ligated to the Sau 3AI site of the truncatedrIFN-αA DNA fragment (100 ng) in a volume of 7 μl at 15° C. for 16 hrs.in the presence of T4 ligase. After ligation, the ligase was inactivatedby incubation at 70° C. for 10 min. and the DNA was digested with 50units of Eco RI and 10 units of Pst I for 2 hrs. at 37° C. in 20 μl inorder to regenerate the "sticky ends". The resultant 865 base pair dsDNAfragment, which contained the coding sequence for rIFN-αA (Gly 1), hadan Eco RI site at the 5' end and a Pst I site at the 3' end of thecoding region. The fragment was purified through a 1.5% Agarose gel andabout 50 ng were recovered for cloning.

(b) Preparation of a Replicable Plasmidic Expression Vehicle forProducing rIFN-αA (Gly 1)

FIG. 4 is a schematic representation of the procedure which was employedto prepare the expression vehicle. We employed plasmid pRC 23 as theexpression vector to prepare the replicable plasmidic expression vehiclefor producing rIFN-αA (Gly 1). As previously indicated, this plasmid wasderived from pBR 322 and contained a P_(L) promoter-operator. Since pRC23 was designed to accept genes on an Eco RI restriction site, it wasnecessary to convert the 3' Pst I site of the 865 base pair fragment toan Eco RI site. In order to have a sufficient amount of the fragment toperform the modification, we first cloned the 865 base pair fragment,using plasmid pBR 322, which contains a tetracycline resistance gene, asa cloning vector. We digested 500 ng of pBR 322 to completion at 37° C.with 10 units each of Eco RI and Pst I in a 20 μl reaction. Thelinearized vector thus produced was purified through a 1.0% Agarose gel.The 865 base pair fragment encoding rIFN-αA (Gly 1) (50 ng) was ligatedwith 100 ng of the linearized pBR 322 using T4 ligase in 10 μl for 8hrs. at 15° C. After ligation, the mixture was incubated at 70° C. for10 min. and used to transform a competent MC 1061 strain of E. colicells. The cells were plated out on LB agar containing 10 μg/ml oftetracycline and the tetracycline resistant colonies obtained fromincubation at 37° C. were screened for the presence of the plasmid pBR322/rIFN-αA (Gly 1). Plasmid screening was performed by the alkalinelysis procedure of Birnboim, H. C. and Doly, J., Nucleic Acids Res. 7,1513 (1979).

Synthetic oligodeoxynucleotides having the sequences ##STR6## wereprepared by the procedure described in Miyoshi, et al., Nucleic AcidsRes. 8, 5507-5517 (1980) and phosphorylated with ATP in the presence ofpolynucleotide kinase at the 5' ends for ligation.

The plasmid pBR 322/rIFN-αA (Gly 1) (200 ng), which contained the 865base pair insert encoding rIFN-αA (Gly 1), was digested to completionwith 20 units of Pst I at 37° C. for 1 hr. and the linearized vector waspurified through a 1.0% Agarose gel and recovered from the gel. Thesynthetic oligodeoxynucleotides (50 ng each) were ligated to 200 ng ofthe linearized vector in a volume of 5 μl at 15° C. for 16 hrs. Afterligase inactivation, the resultant dsDNA was digested with 50 units ofEco RI for 3 hrs. at 37° C. An 869 base pair fragment encoding rIFN-αA(Gly 1) and terminating in an Eco RI site at either end, was purified on1.0% Agarose gel and recovered for cloning into the Eco RI site of pRC23.

About 500 ng of pRC 23 was digested to completion with 10 units of EcoRI at 37° C. for 1 hr. in 20 μl. There was added 1 μg of calf intestinalalkaline phosphatase to dephosporylate the Eco RI ends of the plasmidand incubation was continued for an additional 30 min. The reaction wasterminated by heating at 68° C. for 10 min. and the linearized vectorwas purified through a 1.0% Agarose gel and recovered. The linearizedvector (100 ng) was ligated with 50 ng of the 869 base pair fragmentencoding rIFN-αA (Gly 1) in 10 μl at 15° C. for 10 hrs. to produce thereplicable plasmidic expression vehicle pRC 23/rIFN-αA (Gly 1).

(c) Preparation of Transformant Containing pRC 23/rIFN-αA (Gly 1)

The ligation mixture was used to transform an RR1 strain of E. colicells which contained the compatible plasmid pRK 248 cIts as describedby Bernard, H. U. and Helinski, D. R, Methods in Enzymology, 18, 482.The compatible plasmid encodes the production of the repressor proteinwhich recognizes and binds the operator portion of the P_(L)promoter-operator on pRC 23. The E. coli cells were transformed at 4° C.for 30 min. in the presence of 50 mmol CaCl₂ The cells were plated outat 30° C. on LB agar containing ampicillin (50 μg/ml). The pRC 23plasmid contains a gene for ampicillin resistance. After incubation theampicillin resistant colonies were selected and screened for thepresence of the plasmid pRC 23/rIFN-αA (Gly 1) with the gene inserted inthe proper orientation relative to the P_(L) promoter.

(d) Expression and Purification of rIFN-αA (Gly 1)

A colony of transformants containing the plasmid pRC 23/rIFN-αA (Gly 1)and the compatible plasmid was grown up in 2 ml of LB agar containingampicillin (50 μg/ml) at a temperature which was not allowed to exceed30° C. When the OD₆₀₀ of the culture reached 0.6, the temperature wasraised to 42° C. to inactivate the repressor protein and initiateexpression. After 2 hrs. at 42° C. the cells were harvested and lysed in50 μl of 7M Guanidine-HCl at 0° C. for 10 min. Extracts were centrifugedfor 5 min. at 12,000 g. The supernatants were diluted 1:100 forantiviral assay.

The rIFN-αA (Gly 1) was extracted from E. coli cell paste using anextraction buffer containing 2M Guanidine-HCl, 2% Triton×100, 0.1MTris-Cl, pH 7.5, for 2 hrs. at 4° C. The extraction mixture was dilutedfive-fold with cold distilled water, centrifuged at 10,000×g. for 1 hr.and purified on a 1×2-cm immunoaffinity chromatography column. Theimmunoaffinity column was packed with 1.0 ml of Agarose gel to whichthere was covalently bound approximately 13 mg of a monoclonal antibodyto rIFN-αA. Preparation of the monoclonal antibody, identified as Li-8,is described by Staehelin et al. in J. Biol. Chem., 256, 9750 (1981).

The rIFN-αA/Gly 1-containing extraction buffer (20 ml) was loaded ontothe column, which was operated at a flow rate of 1.0 ml/min. or less.The column was then sequentially washed with 5 bed-volumes of thesolutions containing

1. 0.286M Guanidine-HCl; 0.286% Triton x-100; 0.1M Tris-Cl, pH 7.5

2. 0.5M NaCl; 0.2% Triton x-100; 0.025M Tris-Cl, pH 7.5

3. 1.0M sodium thiocyanate; 0.1% Triton x-100; 0.025M Tris-Cl, pH 7.5

4. 0.15M NaCl; 0.1% Triton x-100.

The rIFN-αA (Gly 1) was then eluted from the column using an elutionsolvent of 0.2M acetic acid, 0.15M NaCl and 0.1% Triton x-100 at pH 2.5.

The eluate from the immunoaffinity chromatography column was tested forantiviral activity against vesicular stomatitis virus using thecytopathic effect inhibition test described in Familletti, et al.,supra. The specific activity of the eluate containing the rIFN-αA(Gly 1) was 2×10⁸ units/mg of protein, which corresponds to that ofrIFN-αA.

Prior to raising the temperature to 42° C. to initiate expression, analiquot of the culture containing the transformants was set aside andstored in glycerol at 30° C. This aliquot was then grown up in a10-liter fermentor containing LB agar containing ampicillin 50 μg/ml ata temperature not exceeding 30° C. until the OD₆₀₀ reached between 4 and5. The temperature was then raised to 42° C. to initiate expression.After the OD₆₀₀ reached about 12 (2-3 hrs), the rIFN-αA (Gly 1) wasrecovered and purified in a manner similar to that described above forthe 2-ml culture. The purified rIFN-αA (Gly 1) obtained from the10-liter culture had an antiviral activity on MDBK cells of 3.3×10⁸(±0.76×10⁸) units/mg of protein.

The purified rIFN-αA (Gly 1) eluate from the immunoaffinitychromatography column was electrophoresced on sodium dodecylsulfatepolyacrylamide gel under non-reducing conditions. FIG. 5 is a photographof the gel. Track 1, which is a sample of rIFN-αA, displayed two bandscorresponding to a slow-moving monomer and a fast-moving monomer, aswell as dimer, trimer and tetramer. Track 2, which is a sample ofrIFN-αA (Gly 1), displayed only slow-moving monomer and a small amountof dimer. Track 3, which represents rIFN-αA (Gly 1) which has beenneutralized to pH 7.0 shows an increase in the dimer from due topH-dependent dusulfide bond formation between cysteines at position 98.Track 4 represents a sample of the material from track 3 reduced withβ-mercaptoethanol and demonstrates that the dimer is a result ofdisulfide bond formation.

EXAMPLE 2 Preparation of rIFN-αA (Gly 1/Ser 98)

(a) Preparation of dsDNA Encoding rIFN-αA (Gly 1/Ser 98)

We prepared the dsDNA encoding rIFN-αA (Gly 1/Ser 98) by takingadvantage of a Pvu II site upstream of the nucleotide triplet in therIFN-αA (Gly 1) gene encoding the cysteine at position 98 and a Hinf Isite downstream of the nucleotide triplet. The Pvu II site and the HinfI site are separated by 48 bases on the coding strand.

Referring to FIG. 3, we employed as a starting material pRC 23/rIFN-αA(Gly 1) which contained the 869 base pair insert of Example 1 encodingrIFN-αA (Gly 1). We cleaved this 869 base pair segment out of pRC 23 bydigesting to completion with 20 units of Eco-RI and 20 units of Pvu IIfor 2 hrs. at 37° C. in 20 μl. The 869 base pair segment was thuscleaved out of pRC 23/rIFN-αA (Gly 1) at the Eco RI sites and was itselfcleaved at the Pvu II site just upstream of the nucleotide tripletencoding cysteine at position 98 in two fragments--a 273 base pairfragment containing the 5' end of the rIFN-αA (Gly 1) gene and a 596base pair segment containing the 3' end of the gene including thenucleotide triplet encoding cysteine at position 98. The 596 base pairfragment was partially digested with 5 units of Hinf I at 37° C. for 5min. in 20 μl. Hinf I was inactivated immediately at 70° C. for 10 min.A 548 base pair Hinf I-Eco RI fragment containing the 3' end of therIFN-αA (Gly 1) gene was isolated and purified on 1.5% Agarose geleliminating a 48 base pair fragment containing the triplet encoding thecysteine at position 98.

Using the procedure of Miyoshi, supra, two double stranded syntheticoligodeoxynucleotides having a complementary overlapping sequence at the3' end of the coding strand of the first oligodeoxynucleotide and the 5'end of the coding strand of the second oligodeoxynucleotide wereprepared. When ligated at the complementary sequence, these twooligodeoxynucleotides produced a 48 base pair insert which was identicalto the 48 base pair fragment cleaved out of the rIFN A (Gly 1) geneexcept that the nucleotide triplet encoding cysteine at position 98 wasreplaced by an AGC nucleotide triplet encoding serine.

The two double stranded synthetic oligodeoxynucleotides had thefollowing nucleotide sequences: ##STR7##

Prior to annealing the individual strands together to produce the twodouble strands, the 5' ends were phosphorylated with ATP usingpolynucleotide kinase to allow ligation. Block I (50 ng) was ligated tothe Pvu II site of the 273 base pair cleavage fragment at the 5' end ofthe rIFN-αA (Gly 1) gene (300 ng) for 16 hrs. at 15° C. in a 7 μlreaction. Block II (50 ng) was then ligated to the Hinf I site of the548 base pair cleavage fragment at the 3' end of the rIFN-αA (Gly 1)gene (100 ng) for 16 hrs. at 15° C. in a 7 μl reaction. The two dsDNAfragments produced by these ligations were then ligated to each other atthe 9 base pair complementary overlapping portions of Block I and BlockII for 16 hrs. at 15° C. The resultant 869 base pair dsDNA encodingrIFN-αA (Gly 1/Ser 98) was isolated and purified on a 1.5% Agarose gel.The dsDNA had an Eco RI recognition site at either end.

(b) Preparation of a Replicable Plasmidic Expression Vehicle forProducing rIFN-αA (Gly 1/Ser 98)

FIG. 6 is a schematic representation of the procedure which was employedto prepare the expression vehicle. Plasmid pRC 23 (500 ng) was digestedto completion with 10 units of Eco RI at 37° C. for 1 hr. in 20 μl.There was then added 1 μg of calf intestinal alkaline phosphatase andincubation was continued an additional 30 min. The reaction wasterminated by heating at 68° C. for 10 min. and the linearized vectorwas purified through a 1.5% Agarose gel and recovered. The linearizedvector (100 ng) was ligated with 50 ng of the 869 base pair fragmentencoding rIFN-αA (Gly 1/Ser 98) in 7 μl for 10 hrs. at 15° C. to producethe replicable plasmidic expression vehicle pRC 23/rIFN-αA (Gly 1/Ser98).

(c) Preparation of Transformant Containing pRC 23/rIFN-αA (Gly 1/Ser 98)

The ligation mixture was used to transform an RR1 strain of E. colicontaining the compatible plasmid pRK 248 cIts, encoding the productionof the temperature-sensitive repressor protein which recognizes andbinds the operator sequence of P_(L) promoter-operator on pRC 23. The E.coli cells were transformed at 4° C. for 20 min. in the presence of 50mmol CaCl₂. Ampicillin resistant colonies resulting from overnightincubation at 30° C. on LB plates were selected and inoculated into 2-mlcultures of LB agar containing ampicillin (50 μg/ml). Coloniescontaining the recombinant plasmid pRC 23/rIFN-αA (Gly 1/Ser 98) withthe gene inserted in the proper orientation relative to the P_(L)promoter were selected by screening with the alkaline lysis method(Birnboim, supra).

(d) Expression and Purification of rIFN-αA (Gly 1/Ser 98)

A colony of transformants containing the plasmid pRC 23/rIFN-αA (Gly1/Ser 98) and the compatible plasmid was grown up in 10 liters of LBagar and ampicillin (50 μg/ml) at a temperature which was not allowed toexceed 30° C. When the OD₆₀₀ of the culture reached 0.6, the temperaturewas raised to 42° C. to inactivate the repressor protein and initiateexpression. After 2 hrs. at 42° C. the cells were harvested and lysed.

The rIFN-αA (Gly 1/Ser 98) was extracted from E. coli cell paste using 4volumes of the buffer of Example 1(d) and purified on a 1.6×5.0-cmimmunoaffinity chromatography column. The immunoaffinity chromatographycolumn was packed with 10 ml of Agarose gel to which there wascovalently bound approximately 130 mg of a monoclonal antibody torIFN-αA. Preparation of the monoclonal antibody, identified as Li-8, isdescribed by Staehelin et al. in J. Biol. Chem., 256, 9750 (1981).

The rIFN-αA/(Gly 1/Ser 98)-containing extraction buffer (5,000 ml) wasloaded onto the column, which was operated at a flow rate of 5.0 ml/min.or less. The column was then sequentially washed with 5 bed-volumes ofthe same washing solutions employed in Example 1(d). The rIFN-αA (Gly1/Ser 98) was then eluted from the column using the same elution solventas in Example 1(d).

The eluate from the immunoaffinity chromatography column was tested forantiviral activity against vesicular stomatitis virus using thecytopathic effect inhibition test described in Familletti, supra. Thespecific activity of the eluate containing the rIFN-αA (Gly 1/Ser 98)was 2×10⁸ units/mg of protein, which corresponds to that of rIFN-αA.

The purified rIFN-αA (Gly 1/Ser 98) eluate from the immunoaffinitychromatography column was electrophoresced on sodium dodecylsulfatepolyacrylamide gel under non reducing conditions. FIG. 7 is a photographof the gel. The sample of rIFN-αA (Gly 1/Ser 98), displayed a singleband of monomeric interferon, whereas Track 2, which is a sample ofrIFN-αA, displayed bands corresponding to monomer, dimer, trimer andhigher oligomers.

We claim:
 1. An antiviral composition comprising a biologically activerecombinant or hybrid recombinant human IFN-α in which at least onecysteine residue at position 1, 98, 99 or 100 has been replaced by anamino acid residue that is incapable of forming a disulfide bond, and apharmaceutically acceptable carrier vehicle.
 2. The antiviralcomposition of claim 1 in which the IFN-α is a recombinant human IFN-αAin which at least one cysteine residue at position 1 or 98 has beenreplaced.
 3. A double-stranded DNA comprising a coding strand and acomplementary strand, which coding strand comprises a series ofnucleotide triplets encoding a biologically active recombinant or hybridrecombinant human IFN-α in which at least one cysteine residue atposition 1, 98, 99 or 100 has been replaced by an amino acid residuethat is incapable of forming a disulfide bond.
 4. The double-strandedDNA of claim 3 in which the coding strand, reading from the 5' end,comprises the sequence ##STR8## wherein X and Y encode cysteine or anamino acid residue that is incapable of forming a disulfide bond.
 5. Thedouble-stranded DNA of claim 4 in which X encodes a glycine residue. 6.The double-stranded DNA of claim 4 in which Y encodes a serine residue.7. The double-stranded DNA of claim 4 in which X is the nucleotidetriplet -GGC-.
 8. The double-stranded DNA of claim 4 in which Y is thenucleotide triplet -AGC-.
 9. A replicable plasmidic expression vehiclecomprising:(a) a replicon; (b) a promoter-operator sequence; (c) a DNAsequence encoding a ribosome binding site; (d) a DNA sequence, in phasewith said promoter-operator sequence, comprising a translationinitiation signal, a sequence encoding a biologically active recombinantor hybrid recombinant human IFN-α in which at least one cysteine residueat position 1, 98, 99 or 100 has been replaced by an amino acid residuethat is incapable of forming a disulfide bond, and a translationtermination signal.
 10. The replicable plasmidic expression vehicle ofclaim 9 in which the IFN-α encoded is a recombinant human IFN-αA inwhich at least one cysteine residue at position 1 or 98 has beenreplaced.
 11. The replicable plasmidic expression vehicle of claim 9 inwhich the IFN-α encoded is a recombinant IFN-αD or a hybrid recombinantIFN-α having an IFN-αD amino acid subsequence, which interferonsnormally contain a cysteine residue at position 87, in which suchresidue has been replaced by a serine residue.
 12. The replicableplasmidic expression vehicle of claim 9 in which the sequence encodingthe IFN-α, reading from the 5' end, comprises the sequence ##STR9##wherein X and Y encode cysteine or an amino acid residue that isincapable of forming a disulfide bond.
 13. The replicable plasmidicexpression vehicle of claim 12 in which X encodes a glycine residue. 14.The replicable plasmidic expression vehicle of claim 13 in which Yencodes a serine residue.
 15. The replicable plasmidic expressionvehicle of claim 12 in which X is the nucleotide triplet -GGC-.
 16. Thereplicable plasmidic expression vehicle of claim 12 in which Y is thenucleotide triplet -AGC-.
 17. A microorganism containing the replicableplasmidic expression vehicle of claim 9.